Effect of 6-Thioguanine and Methyl-6-Thioguanine on stability of DNA duplexes

6-Thioguanine (65G) has been used for the past thirty years as an anti-cancer agent in maintenance chemotherapy for childhood acute lymphoblastic leukemia as well as acute myeloid leukemia. Despite its long-standing clinical use, little is understood of the molecular basis of the cytotoxicity associated with ?sG. Research has shown that ?sG is incorporated into DNA during replication after which it may exert its cytotoxicity by inducing rapid mutation or by affecting DNA-protein interactions. 65G has also been shown to impair HIV replication by inhibiting HlV-l reverse transcriptase and to inhibit human telomerase activity. To further understand the structural changes associated with DNA duplexes containing 65G and its metabolite methyl-6-thioguanine (Me65G), we have studied and compared the thermodynamic properties of the 11 base pair duplex dCCGTTAGATGCC).CGGCATCTAAGC) to duplexes in which the central G residue is replaced by either 65G or Me65G using temperature dependent UV-Vis experiments. Results suggest that duplexes with 65G are very similar in stability to those containing normal G while the duplexes with Me65G are considerably destabilized compared to G or 65G

Operational research in low-income countries: what, why, and how?

Operational research is increasingly being discussed at institutional meetings, donor forums, and scientific conferences, but limited published information exists on its role from a disease-control and programme perspective. We suggest a definition of operational research, clarify its relevance to infectious-disease control programmes, and describe some of the enabling factors and challenges for its integration into programme settings. Particularly in areas where the disease burden is high and resources and time are limited, investment in operational research and promotion of a culture of inquiry are needed so that health care can become more efficient. Thus, research capacity needs to be developed, specific resources allocated, and different stakeholders (academic institutions, national programme managers, and non-governmental organisations) brought together in promoting operational research

Treatment outcomes of patients on anti-retrovirals after six months of treatment, Khami Clinic, Bulawayo, Zimbabwe

A CAJM review of HIV/AIDS treatment of infected patients on medication after 6 months of administering anti-retrovirals.It was in 1985 that the first case of HIV tested positive in Zimbabwe. The AIDS epidemic has grown since then to become one of the most serious public health challenges to ever face the nation. According to the 2003 HIV estimates, 24,6% of adults aged 15 to 49 years were infected. Whilst they cannot cure HIV/AIDS, treatment of HIV with Highly Active Antiretroviral Therapy (HAART) can transform the natural course of HIV infection by reducing morbidity and mortality as has been observed in many industrialized countries. It is recommended for patients with symptomatic AIDS, WHO Adult Stage IV and advanced Stage III irrespective of the CD4 cell count or total lymphocyte count

Health effects of agrochemicals among farm workers in commercial farms of Kwekwe district, Zimbabwe

Introduction: Farm workers are at a very high risk of occupational diseases due to exposure to pesticides resulting from inadequate education, training and safety systems. The farm worker spends a lot of time exposed to these harmful agrochemicals. Numerous acute cases with symptoms typical of agrochemical exposure were reported from the commercial farms. We assessed the health effects of agrochemicals in farm workers in commercial farms of Kwekwe District (Zimbabwe), in 2006. Methods: An analytical cross sectional study was conducted amongst a sample of 246 farm workers who handled agrochemicals when discharging their duties in the commercial farms. Plasma cholinesterase activity in blood specimens obtained from farm workers was measured using spectrophotometry to establish levels of poisoning by organophosphate and/or carbamates. Information on the knowledge, attitudes and practices of farm workers on agrochemicals use was collected using a pre-tested interviewer administered questionnaire. Bivariate and multivariate analyses were conducted to determine factors that were associated with abnormal cholinesterase activity. Results: The prevalence of organophosphate poisoning, indicated by cholinesterase activity of 75% or less, was 24.1%. The median period of exposure to agrochemicals was 3 years (Q1: = 1 year, Q3: = 7 years). Ninety eight (41.5%) farm workers knew the triangle colour code for the most dangerous agrochemicals. Not being provided with personal protective equipment (OR 2.00; 95% CI: 1.07 – 3.68) and lack of knowledge of the triangle colour code for most dangerous agro-chemicals (OR 2.02; 95% CI: 1.02 – 4.03) were significantly associated with abnormal cholinesterase activity. Conclusion: There was organophosphate poisoning in the commercial farms. Factors that were significantly associated with the poisoning were lack of protective clothing and lack of knowledge of the triangle colour code for most dangerous agrochemicals. We recommended intensive health education and training of farm workers on the use of agrochemicals, provision of adequate and proper personal protective equipment as mitigation measures to this problem.Key words: Organophosphates, cholinesterase activity, pesticide poisoning, agrochemicals, farm worker

New Policies, New Technologies: Modelling the Potential for Improved Smear Microscopy Services in Malawi

Background To quantify the likely impact of recent WHO policy recommendations regarding smear microscopy and the introduction of appropriate low-cost fluorescence microscopy on a) case detection and b) laboratory workload.Methodology/Principal Findings An audit of the laboratory register in an urban hospital, Lilongwe, Malawi, and the application of a simple modelling framework. The adoption of the new definition of a smear-positive case could directly increase case detection by up to 28%. Examining Ziehl-Neelsen (ZN) sputum smears for up to 10 minutes before declaring them negative has previously been shown to increase case detection (over and above that gained by the adoption of the new case definition) by 70% compared with examination times in routine practice. Three times the number of staff would be required to adequately examine the current workload of smears using ZN microscopy. Through implementing new policy recommendations and LED-based fluorescence microscopy the current laboratory staff complement could investigate the same number of patients, examining auramine-stained smears to an extent that is equivalent to a 10 minutes ZN smear examination.Conclusions/Significance Combined implementation of the new WHO recommendations on smear microscopy and LED-based fluorescence microscopy could result in substantial increases in smear positive case-detection using existing human resources and minimal additional equipment

Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS) Are Active In Vivo in Anemic Baboons and Transgenic Mice

We thank Sarah Haigh, Ada Kane, Nicole Reuter, David Carey, and Marilyn Perry Carey for dedicated and expert technical assistance and Cloret Carl for assistance with preparation of the manuscript.This work was supported by grants from the National Institutes of Health, R01 DK-52962, (SPP, Boston University), R41 HL-105816 (SPP, Phoenicia BioSciences), and R42 HL-110727 (Phoenicia BioSciences), 2 P40 ODO010988-16 (GLW, University of Oklahoma) and UL1-TR000157 (RFW, University of Oklahoma). SMN was supported by P50 HL-118006. The funders had no role in study design, data collection or analysis, decision to publish, or preparation of the manuscript.High-level fetal (γ) globin expression ameliorates clinical severity of the beta (β) hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies.Yeshttp://www.plosone.org/static/editorial#pee

5-Azacytidine Induces Human Fetal Hemoglobin Production through Activation of the p38 MAPK and Integrated Stress Response Signaling Pathways

Abstract Pharmacologic induction of fetal hemoglobin (HbF) has the potential to improve the health and quality of life of people with β-thalassemia and sickle cell disease. 5-Azacytidine (5-Aza) is a key lead compound as it, and the related drug decitabine, are active in most β-thal and SCD patients including those resistant to butyrate and because they have been shown to produce clinical benefits for selected patients. However, these drugs must be administered by injection, they depress blood counts and they are known to alter DNA structure and cause changes in genome-wide DNA methylation. Efforts to design improved inducing agents have been limited by an incomplete understanding of the mechanisms underlying pharmacologic induction of HbF. Current theories include proposals that 5-Aza and decitabine induce increased γ-globin gene expression by altering the kinetics of erythroid differentiation or by decreasing γ-globin promoter DNA methylation. We recently provided evidence that the primary action 5-Aza is not through either of these mechanisms. These results, and data from the literature, have led us to propose a new model of HbF induction based on activation of cell stress signaling pathways. To begin to evaluate this model, we used a human in vitro CD34 erythroid differentiation system to test the hypotheses that the p38 MAPK stress signaling pathway was involved in 5-Aza induction of HbF. Quantitative RT-PCR, Western blotting and HPLC were used to assess mRNA, protein and Hb levels. Our results showed that 500 nM 5-Aza causes p38 MAPK pathway activation as evidenced by phosphorylation of p38 MAPK, the downstream kinase MK2 and the downstream target Hsp27. The p38 MAPK inhibitor SB203580 (SB) prevented pathway activation and suppressed both baseline and 5-Aza-induced γ-globin mRNA production. On day +13 of differentiation, relative γ-globin mRNA levels were 1.00 (untreated), 0.40 (SB alone), 2.18 (5-Aza alone) and 0.46 (5-Aza + SB) All values were p&amp;lt;0.05 vs. control. HbF levels at the end of differentiation were 2.1% (untreated), 2.2% (SB alone), 24.1% (5-Aza) and 10.7% (5-Aza + SB). These results indicate that the p38 MAPK pathway is activated by 5-Aza and that inhibition of this pathway suppresses 5-Aza-induced increases in γ-globin mRNA and HbF production. However, the fact that SB did not completely inhibit HbF production suggested other pathways might be involved. The Integrated Stress Response (ISR) pathway (also known as the Unfolded Protein Response pathway) responds to a variety of stresses with phosphorylation of translation factor eIF2α. While this initially inhibits translation of most proteins, production of the ATF4 transcription factor is increased and it mediates a secondary transcriptional response. This pathway is known to be activated in differentiating erythroid cells by inadequate heme and other stresses. 500 nM 5-Aza caused pathway activation as evidenced by phosphorylation of both heme-regulated eIF2α kinase (HRI) and eIF2α and by increased levels of ATF4. Expression of a dominant-negative ATF4 protein inhibited 5-Aza induction of γ-globin mRNA by more than 50%. Independent activation of the ISR pathway by L-azetidine-2-carboxylic acid (a proline analogue that causes protein miss-folding), induced γ-globin mRNA and HbF to levels equivalent to those seen with 5-Aza. 5-Aza also alters the polysome profiles of γ and β-globin mRNA in differentiating human cells (50% increase in polysome-associated γ-globin mRNA, p=0.02; 55% decrease in β-globin mRNA, p&amp;lt;0.01). Taken together, these results suggest that 5-Aza induces HbF production through activation of the p38 MAPK and ISR cell signaling pathways and that this induction involves both transcriptional and translational effects. The identification of cell signaling pathways involved in HbF induction opens the possibility of future targeted drug development.</jats:p